Capillary chromatographic separation methods are preferably performed in fused silica (FS) tubing with internal diameters ranging from 5-530 .mu.m. Such tubing consists of a silica (SiO.sub.2) glass drawn at high temperature (1300.degree. C.) from a quartz preform provided with a protective outside layer from polyimide or aluminum. Robustness, tensile strength, high pressure resistance and bend stability are favorable mechanical properties of fused silica tubing. High chemical purity and well defined surface of the tubing provides in most cases low interaction with solutes and leads to optimum separation in many applications.
In U.S. Pat. No. 4,293,415 Dandeneau et al. describe the usage of a fused silica capillary, which may have wall coatings on the inside surface to stimulate specific interactions and/or further minimize secondary undesired solute/surface interactions, for open tubular capillary gas chromatography (CGC) and open tubular supercritical fluid chromatography (SFC). Jorgenson et al. have demonstrated that such capillaries are also ideally suited for the newer technique of capillary electrophoresis (CE).
It has been demonstrated that FS tubing can also be used for capillary separations performed in a packed bed, such as SFC, micro high performance liquid chromatography, and capillary electrochromatography (CEC). The mechanical properties of fused silica capillaries suffice to withstand the high pressure that occurs either when packing the tubing with small particles using a high pressure filtration technique or when operating the column especially in high performance liquid chromatography mode.
The main problem in packing fused silica or other tubing with small inner diameter is that the packing material in the column bed needs to be retained in the tubing; otherwise hydraulic or electrical forces drive the particles out of the capillary column. In a conventional high performance liquid chromatography column the packed bed is typically kept in place under the high pressure that is applied (up to 400 bar) by terminating plates or sieves, called frits, that are porous to the liquid but too narrow for the particles to move through. Because these frits need to be firmly attached to the packed bed, a fitting is needed which compresses the frit to the bed and at the same time resists the high pressure. In conventional high performance liquid chromatography columns, stainless steel fittings are used that are clamped to the column tube outside.
Due to the narrow outer diameter of the fused silica capillary tubing, typically 0.350 mm, and the small volumes involved in the separation, it is not very well possible to use external fittings even if they are reduced in size accordingly.
Several groups therefore have pursued the principal approach to immobilize part of the packed bed in the capillary by chemical means. E.g. Heman Cortez et al. in U.S. Pat. No. 4,793,920 describe the usage of KaSil (potassium silicate) to form a porous ceramic frit in the fused silica tube which will retain the small particles during column packing. Columns with frit terminators made in this way have internal diameters typically in the 180-530 .mu.m range and have been used in SFC preferably.
In micro high performance liquid chromatography and the new field of capillary electrochromatography (CEC) narrower columns--interior diameter &lt;200 .mu.m--are used. In this field, several groups have pursued other approaches to form such a frit. In U.S. Pat. No. 5,246,577 Fuchs et al. bring fusable glass beads, 1-50 .mu.m diameter into the fused silica capillary tube and melt these together under electrical sparking.
Unmodified silica particles 3-40 .mu.m diameter have been used alternatively. After bringing these into the fused silica capillary tube, particles were glued together by destabilization of a tetraalkoxysilane forming in situ silicic acid binding the particles together.
In recent publications the stationary phase particles have been immobilized directly in the packed bed by application of heat to a zone of the packed fused silica column where the terminating frit needs to be while the column still is at high pressure on the packing apparatus (e.g. Boughtflower et al., Chromatographia 40, 329 (1995), Smith et al., Chromatographia 38, 649 (1994), Rozing et al., LC-GC Magazine, October 1995). It is believed that under these conditions the particles are glued together by the fact that upon heating a small amount of silica dissolves in water forming silicic acid, and that upon cooling the repolymerized silicic acid deposits between the particles. The advantage of this approach is that it does not substantially alter the chemical constitution of the zone that is fritted, that it can be done on the inlet and outlet side without problem, that the length of the fritted zone is well controlled by the dimension of the external heating source used and that the porosity of the bed is unaffected. Photographs e.g. by Boughtflower et al., show that the particle structure is not affected by this treatment and therefore interparticle porosity is maintained.
The main problem with all these approaches is to obtain chemical or physical adhesion of the fritted zone to the inner capillary wall so that the fritted zone has sufficient stability to overcome shrinking and cracking of the bed or fritted zone. It has been observed that after drying out of a packed capillary the frits loose contact to the inner capillary wall and gentle electrical or hydraulic force on the bed suffices to drive out the packing and destroy the column. With all approaches to generate internal frits in a packed capillary column attachment of the fritted zone to the inside wall of the capillary remains a potential problem.